Food Chemistry 134 (2012) 20902096

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Food Chemistry
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Phytochemical prole of a blend of black chokeberry and lemon juice

with cholinesterase inhibitory effect and antioxidant potential
Amadeo Girons-Vilaplana a, Patrcia Valento b, Paula B. Andrade b, Federico Ferreres a,
Diego A. Moreno a,, Cristina Garca-Viguera a
a
b

Department of Food Science and Technology, CEBAS-CSIC, P.O. Box 164, E-30100, Espinardo, Murcia, Spain
REQUIMTE/Laboratrio de Farmacognosia, Departamento de Qumica, Faculdade de Farmcia, Universidade do Porto, R. Anbal Cunha 164, 4050-047 Porto, Portugal

a r t i c l e

i n f o

Article history:
Received 16 January 2012
Received in revised form 9 March 2012
Accepted 3 April 2012
Available online 13 April 2012
Keywords:
Lemon
Black chokeberry
Cholinesterase
Antioxidant
Neurodegenerative

a b s t r a c t
In this study, black chokeberry concentrate was added (5% w/v) to lemon juice, since previous reports
suggested potential health benets of this blend. The phytochemical composition, antioxidant capacity
(scavenging of DPPH, superoxide and hydroxyl radicals, and hypochlorous acid), and inhibitory activity
against cholinesterase of the new blend were determined and compared with those of lemon juice and
chokeberry in citric acid (5%). The chokeberry concentrate, rich in cyanidin-glycosides, quercetin derivatives, and 3-O-caffeoylquinic acid, and lemon juice, possessing avones, avanones, quercetin derivates,
and hydroxycinnamic acids, were characterised. The new drink showed a higher antioxidant effect than
the chokeberry or lemon controls for all the tested methods, except for hypochlorous acid, in which
lemon juice displayed higher activity. Both the lemon juice and chokeberry controls inhibited acetylcholinesterase and butyrylcholinesterase, and this effect was increased in the new mixtures. The results of
the different radical scavenging assays indicate that the lemonblack chokeberry (5% w/v) mixture
was more antioxidative than the respective controls separately. Moreover, their inhibition of cholinesterase is of interest regarding neurodegenerative disorders such as Alzheimers disease, Parkinsons disease,
or senile dementia.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
There is increasingly strong scientic evidence that a diet rich in
fruits and vegetables may reduce the risk of suffering different
chronic diseases (Borges, Mullen, & Crozier, 2010). The results obtained with the techniques employed in the past were insufcient
to support this relationship, so it was necessary to examine more
thoroughly the characteristics of the food in order to identify possible metabolites responsible for these preventive effects. Consequently, the current trend is to study foods in order to
demonstrate that this relationship between consumption and prevention is correct. An easy and convenient form of consuming
fruits is in juice form, and one focus of current research is the design of fruit-based beverages rich in phytochemicals and the evaluation of their bioactivity.
Citrus fruits are among the most important horticultural crops,
lemon (Citrus limon (L.) Burm. f.) being the third most important
Citrus crop species (Gonzlez-Molina, Domnguez-Perles, Moreno,
& Garca-Viguera, 2010). Several studies have pointed out that
lemon is a rich source of nutrients and phytochemicals, including
Corresponding author. Tel.: +34 968 396304; fax: +34 968 396213.
E-mail address: dmoreno@cebas.csic.es (D.A. Moreno).
0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.04.010

avonoids, citric acid, vitamin C, and minerals (Gonzlez-Molina,

Moreno, & Garca-Viguera, 2008a), which have numerous health
promoting properties (Gonzlez-Molina et al., 2010; Mulero
et al., 2012). For this reason, lemon juice is an interesting food
matrix for designing new beverages, as well as being a suitable
source of value-added products since overproduction and nonmarketable fruits lead to a serious environmental problem of
unused agrowaste on a yearly basis. In this regard, lemon juice
represents an alternative for the conversion of a bioburden into
a food product.
Black chokeberry (Aronia melanocarpa (Michx.) Elliott) is a
natural, rich source of phenolic antioxidants, such as cyanidin
3-O-glycoside anthocyanins (cyanidin 3-O-galactoside, cyanidin
3-O-glucoside, cyanidin 3-O-arabinoside, and cyanidin 3-O-xyloside) (Gonzlez-Molina, Moreno, & Garca-Viguera, 2008b), quercetin derivatives (Bermdez-Soto & Toms-Barbern, 2004),
hydroxycinnamic acids (Zheng & Wang, 2003), and, in smaller
amounts, vitamin C (Benvenuti, Pellati, Melegari, & Bertelli, 2004;
Skupien & Ozmianski, 2007). Many reports have suggested its
anti-proliferative effects against cancer cells (Lala et al., 2006), as
well as antimutagenic (Gasiorowski, Szyba, Brokos, Kolaczynska,
Jankowiak-Wlodarczyk & Oszmianski, 1997), hepatoprotective
(Kowalczyk et al., 2004), cardioprotective (Hellstrm et al., 2010),

A. Girons-Vilaplana et al. / Food Chemistry 134 (2012) 20902096

and antidiabetic activities (Valcheva-Kuzmanova, Kuzmanov, Tancheva, & Belcheva, 2007). Moreover, a series of papers reported
the antioxidant properties of black chokeberry extracts or their
phenolic constituents, using various, well established in vitro and
in vivo models for direct antioxidant capacity (Bermdez-Soto &
Toms-Barbern, 2004; Rop et al., 2010; Zheng & Wang, 2003), as
well as their protective effects against oxidative stress (Kedzierska,
Olas, Wachowicz, Stochmal, Oleszek & Erler, 2011). Recently, the
neuroprotective effects of cyanidin-3-O-glycosides, commonly
present in black chokeberry, have been tested in mice (Min et al.,
2011).
In the regulation of cognitive functions, the central cholinergic
system is considered to be the most important neurotransmitter involved (Mukherjee, Kumar, Mal, & Houghton, 2007). In addition,
cholinesterases, such as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), are key enzymes that play important roles in
cholinergic transmission, by hydrolysing the neurotransmitter acetylcholine (Vinholes et al., 2011). Cholinergic neuronal loss in the
hippocampal area is the major feature of Alzheimers disease
(AD), senile dementia, ataxia, myasthenia gravis, and Parkinsons
disease, and inhibitors of AChE and BuChE are the pharmacological
treatment used for these diseases (Mukherjee et al., 2007). These
drugs have adverse side-effects, like gastrointestinal disturbances,
nausea, vomiting, and diarrhoea, as well as problems of bioavailability. Because of this, researchers are seeking natural AChE and
BuChE inhibitors with a better safety prole. A wide range of plant
compounds with this inhibitory activity have been found, mainly
alkaloids (Mukherjee et al., 2007), xanthones (Brhlmann, Marston,
Hostettmann, Carrupt, & Testa, 2004), and avonols, such as quercetin (Khan et al., 2009), among others. To the best of our
knowledge, no previous work has been published on lemon or black
chokeberry compounds as inhibitors of cholinesterases.
Following previous research on lemon juice enriched with black
chokeberry concentrate (Gonzlez-Molina et al., 2008b), the aims
of this work were to perform a deeper phytochemical characterisation of this new blend, to extend our knowledge of its antioxidant
capacity, and to evaluate its potential with respect to inhibition of
cholinesterases, for future nutrition and health uses.
2. Material and methods
2.1. Chemicals
The reagents used were commercially available: 2,2-diphenyl1-picrylhidracyl radical (DPPH), b-nicotinamide adenine dinucleotide (NADH), phenazine methosulfate (PMS), nitrotetrazolium blue
chloride (NBT), trizina hydrochloride, bovine albumin, sodium
chloride, acetylcholinesterase from electric eel, butyrylcholinesterase from equine serum, acetylthiocholine iodide, S-butyrylthiocholine chloride, 5,50 -dithiobis(2-nitrobenzoic acid) (DTNB), sodium
borohydride, sodium hypochlorite solution, ferrium chloride (45%
solution), 2-deoxy-D-ribose, and 2-thiobarbituric acid were obtained from SigmaAldrich (Steinheim, Germany); potassium dihydrogen phosphate, ethylenediaminetetraacetic acid (EDTA),
hydrogen peroxide (30%), and trichloroacetic acid were purchased
from Merck (Darmstadt, Germany); magnesium chloride hexahydrate and ascorbic acid were bought from Fluka Chemika (NeuUlm, Switzerland). Ultrapure water was produced using a Millipore
water purication system.
2.2. Samples
Black chokeberry concentrate (62.3 Brix; pH 3.58) was provided by Juver Alimentacin S.A. (Churra, Murcia, Spain). Lemon
juice was obtained, from Fino lemons freshly collected from the

2091

CEBAS-CSIC experimental farm (La Matanza, Santomera, Murcia,

SE Spain; 3860 1400 N, 110 5900 W), using a domestic squeezer (Citromatic, Braun Espaola S.A., Barcelona, Spain). The juice was
stored frozen (20 C) until use.
2.3. Experimental design
Black chokeberry concentrate was added to a volume of lemon
juice to obtain a nal concentration of 5% w/v (g/ml) in the beverage. In addition, a control solution using the same proportion in
0.18 M citric acid buffer (pH 2.46) was prepared, to study the activities of the concentrate without lemon. Lemon juice alone was also
assayed (pH 2.14).
The homogenised mixtures and control solutions were centrifuged (7 min at 4000 rpm). The juices were stored frozen
(20 C) until use.
The samples were labelled as follows: L (lemon juice control),
LA (lemon juice plus 5% black chokeberry concentrate), A (5% black
chokeberry concentrate in citric acid buffer).
2.4. HPLC-DAD-ESI/MSn
The chromatographic analyses were carried out on a Luna C18
column (250  4.6 mm, 5 mm particle size; Phenomenex, Maccleseld, UK). Water:formic acid (99:1, v/v) and acetonitrile were used
as the mobile phases A and B, respectively, with a ow rate of 1 ml/
min. The linear gradient started with 8% of solvent B, reaching 15%
at 25 min, 22% at 55 min, and 40% at 60 min, which was maintained up to 70 min. The injection volume was 30 ll. Chromatograms were recorded at 280, 320, 360, and 520 nm for
hydroxycinnamic acids, non-coloured avonoids, and anthocyanins. The HPLC-DAD-ESI/MSn analyses were carried out in an Agilent HPLC 1100 series machine equipped with a photodiode array
detector and a mass detector in series (Agilent Technologies, Waldbronn, Germany). The equipment consisted of a binary pump
(model G1312A), an autosampler (model G1313A), a degasser
(model G1322A), and a photodiode array detector (model
G1315B). The HPLC system was controlled by ChemStation software (Agilent, version 08.03). The mass detector was an ion trap
spectrometer (model G2445A), equipped with an electrospray ionisation interface and controlled by LCMSD software (Agilent, version 4.1). The ionisation conditions were 350 C and 4 kV, for
capillary temperature and voltage, respectively. The nebulizer
pressure and nitrogen ow rate were 65.0 psi and 11 L/min,
respectively. The full-scan mass covered the range from 100 to
m/z 1200. Collision induced fragmentation experiments were performed in the ion trap using helium as the collision gas, with voltage ramping cycles from 0.3 up to 2 V. The mass spectrometry data
were acquired in the positive ionisation mode for anthocyanins
and in the negative ionisation mode for other avonoids. The
MSn was carried out in the automatic mode on the more abundant
fragment ion in MS(n1).
Prior to injection, the samples were centrifuged (12000 rpm,
5 min) and ltered through a PVDF Filter (0.22 lm). Anthocyanins
were quantied as cyanidin 3-O-glucoside at 520 nm, avonols as
quercetin 3-O-rutinoside (rutin) at 360 nm, hydroxycinnamic acids
as 5-O-caffeoylquinic acid at 320 nm, avanones as hesperidin at
280 nm, and avones as diosmin at 360 nm.
2.5. DPPH radical scavenging activity
The antiradical capacity was estimated spectrophotometrically
in a Multiskan Ascent plate reader (Thermo Electron Corporation),
by monitoring the disappearance of DPPH at 515 nm, according to
Oliveira et al. (2010). Three experiments were performed in
triplicate.

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A. Girons-Vilaplana et al. / Food Chemistry 134 (2012) 20902096

2.6. Superoxide radical (O2) scavenging activity

This antiradical activity was determined spectrophotometrically, in a 96-well plate reader, by monitoring the effect of the controls and the new drink on the O2 induced reduction of NBT at
560 nm. Superoxide radicals were generated by the NADH/PMS
system according to a described procedure (Ferreres, Fernandes,
Oliveira, Valento, Pereira & Andrade, 2009). The experiments were
performed in triplicate.
2.7. Hypochlorous acid scavenging activity
The inhibition of the hypochlorous acid-induced oxidation of 5thio-2-nitrobenzoic acid (TNB) to 5,50 -dithiobis(2-nitrobenzoic
acid) was studied according to a described procedure (Valento,
Fernandes, Carvalho, Andrade, Seabra & Bastos, 2002), in a double
beam spectrophotometer (Hekios a, Unicam, Leeds, UK) at 412 nm.
Three experiments were performed in triplicate.
2.8. Hydroxyl radical assay
The deoxyribose method for determining the scavenging effect
of samples on hydroxyl radicals was performed as described before
(Valento et al., 2002), in a double beam spectrophotometer (Hekios a, Unicam, Leeds, UK) programmed in photometric function,
with the wavelength xed at 532 nm. Three experiments were performed in triplicate.
2.9. AChE and BChE inhibitory activity
The inhibition of AChE activity was determined based on Ellmans method, as reported previously (Ferreres, Taveira, Pereira,
Valento, & Andrade, 2010). The absorbance was measured at
405 nm and the rates of reaction were calculated using Ascent Software version 2.6 (Thermo Labsystems Oy). The BuChE inhibition
assay was performed in a similar way, using 25 ll of substrate
(15 mM butyrylthiocholine) and 25 ll of enzyme (0.1 U/ml). Three
experiments were performed in triplicate.
2.10. Statistical analyses
The data shown are mean values (n = 3). All data were subjected
to analysis of variance (ANOVA) and a Multiple Range Test (Tukeys
test), using PASW Statistics 18 software (Somers, New York, USA).
3. Results and discussion
3.1. Phenolic compounds
The HPLC-DAD study of the lemon juice (L) and black chokeberry (A) controls revealed the presence of a wide range of polyphenols. Some of them were recently identied by our group
(Gonzlez-Molina et al., 2008b; Mellisho et al., 2011). However,
additional phenolic compounds have been identied and quantied in black chokeberry, including cyanidin-glycosides (A1A4)
(Table 1), quercetin derivatives (F1F6) (Table 1), and one
hydroxycinnamic acid, which, by UV spectra and m/z, corresponded to 3-O-caffeoylquinic acid (C1) (Table 1). Lemon juice avones, avanones, quercetin derivatives, and hydroxycinnamic acids
were also characterised (Table 1).
In lemon juice control, avones and avanones were the major
avonoids, followed by hydroxycinnamic acids and quercetin
derivatives (Table 2). Flavones were represented mainly by diosmetin 6,8-di-C-glucoside (L2) (55.9%), followed by diosmetin 7-Orutinoside (L4) (36.9%) and apigenin 6,8-di-C-glucoside (L1)

(7.1%) (Table 2). With respect to avanones, eriodictyol 7-O-rutinoside (L10) and hesperetin 7-O-rutinoside (L11) were present at
very similar levels, each compound representing almost one-half
of the total avanones (48.8 and 51.2%, respectively) (Table 2).
Ferulic acid was the predominant hydroxycinnamic acid (L8)
(50.7%), followed by sinapic acid (L9) (33.5%) and 5-O-caffeoylquinic acid (L7) (15.8%) (Table 2). These compounds have been characterised previously in lemon juice (Mellisho et al., 2011).
However, lower concentrations of avanones and higher amounts
of avones were found when compared to previous results for
Fino lemon juice (Gonzlez-Molina et al., 2008a). It is important
to emphasise that the concentration of avonoids in lemons is variable according to the cultivar, maturity stage, or growth conditions (Gonzlez-Molina et al., 2010).
The black chokeberry control had considerable amounts of total
anthocyanins, quercetin derivatives, and 3-O-caffeoylquinic acid
(Table 2). Cyanidin 3-O-galactoside (A1) was the most abundant
anthocyanin (66.7% of the total), followed by cyanidin 3-O-arabinoside (A3) (26.9%) (Table 2). Cyanidin 3-O-glucoside (A2) and
cyanidin 3-O-xyloside (A4) were present in signicantly lower
amounts (3.1% and 3.3%, respectively) (Table 2). Concerning quercetin derivatives, all the compounds identied displayed similar
concentrations. Quercetin 3-O-galactoside (F5) was the most abundant (31%), followed by the pair quercetin 3-O-rhamnosyl-galactoside plus quercetin 3-O-rhamnosyl-glucoside (26.1%) (F3 + F4)
(Table 2).
In relation to the blend (LA), the anthocyanins concentration
provided by the black chokeberry concentrate was similar to that
of the controls (Table 2). The total hydroxycinnamic acids concentration was higher than in the L and A controls (Table 2). Nonetheless, the avonols provided by both lemon and chokeberry, and the
avones and avanones provided by lemon, were lower (Table 2).
It should be noted that in the new blend (LA), the lemon phytochemicals were slightly diluted with respect to the lemon control,
due to the addition (5%) of chokeberry concentrate. Although some
phytochemicals were less abundant in the new beverage, a positive
effect was observed regarding biological activities, probably because the matrix was richer in bioactive compounds (anthocyanins, avonols, hydroxycinnamic acids, avones, and avanones).
In previous research on lemon juice enriched with black chokeberry concentrate (Gonzlez-Molina et al., 2008b), the levels of
bioactive compounds in the controls and the blends were about
one-third of those reported here, as before the addition of the black
chokeberry concentrate, it was reconstituted to obtain the Brix of
a commercial juice; so, the concentration was 1/3 (w/v) (GonzlezMolina et al., 2008b). Therefore, there is potential for the use of
berry concentrates in the food industry to develop new beverages
with increased concentrations of phytochemicals, in addition to
the advantages of using less volume of fresh fruit or fruit products.
3.2. Antioxidant activity
The antioxidant activity of all the samples was measured as the
scavenging of different radicals: DPPH, O2, hypochlorous acid
(HOCl), and hydroxyl (OH). All of these are considered valid assays
for the evaluation of the antioxidant capacity of plants and foods.
The IC50 values were calculated, in order to compare the different
samples and methods (Table 3).
Concerning DPPH, the lemon juice control showed the lowest
activity. The black chokeberry concentrate had a lower IC50; hence,
its addition to lemon juice resulted in a high scavenging activity of
the blend (Table 3). In previous work (Gonzlez-Molina et al.,
2008b), the addition of 5% black chokeberry concentrate to lemon
juice did not increase the antioxidant activity with respect to the
control, in contrast to our study (Table 3). Another, commercial
black chokeberry juice concentrate had higher activity (60 mg

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A. Girons-Vilaplana et al. / Food Chemistry 134 (2012) 20902096

Table 1
Identication of different bioactive compounds present in chokeberry control and lemon juice.
Compounds
Black chokeberry
Anthocyanins (520 nm)
A1 Cyanidin 3-O-galactoside
A2 Cyanidin 3-O-glucoside
A3 Cyanidin 3-O-arabinoside
A4 Cyanidin 3-O-xyloside
Flavonols (360nm)
F1 Quercetin diglucoside
F2 Quercetin pentosilhexoside
F3 Quercetin 3-O-rhamnosyl-galactoside
F4 Quercetin 3-O-rutinoside
F5 Quercetin 3-O-galactoside
F6 Quercetin 3-O-glucoside
Chlorogenic acid derivatives (320 nm)
C1 3- caffeoylquinic acid
LEMON
Flavones (360 nm)
L1 Apigenin 6,8-di-C-glucoside
L2 Diosmetin 6,8-di-C-glucoside
L3 8-C-glucosilchrysoeriol
L4 Diosmetin 7-O-rutinoside
Flavonols (360nm)
L5 Quercetin 3-O-rutinoside-7-O-glucoside
L6 Quercetin 3-O-rutinoside
Hydroxycinnamic acid derivatives (320 nm)
L7 5-caffeoylquinic acid
L8 Ferulic acid
L9 Sinapic acid
Flavanones (280 nm)
L10 Eriodictyol-7-O-rutinoside
L11 Hesperetin-7-O-rutinoside

Rt

[MH]

MSn

hLambdai (max)

17.1
19.3
21.4
26.7

449
449
419
419

287
287
287
287

520
520
520
520

29.2
34.5
38.6
39.1
40.4
41.5

625
595
609
609
463
463

463, 301
433, 301
301
301
301
301

260,
260,
260,
260,
260,
260,

8.9

353

191, 179

330

24.1
28.0
42.1
52.5

593
623
461
607

503, 473
503, 413, 383
300
299

270,
270,
280,
280,

19.7
38.4

771
609

609, 301
301

265, 365
265, 365

16.5
19.1
20.6

353
175
205

191
169
189

330
330
330

34.3
50.1

595
609

287
301

280
280

360
360
360
360
360
360

345
340
350
345

Rt: Retention time in minutes.

[MH]: Ionisation of masses: Positive for anthocyanins, negative for non-coloured avonoids.

Table 2
Quantication (mg/100 ml) of different phenolic compounds of the lemon juice control, chokeberry control, and new blend.
Compounds
Anthocyanins (520 nm)
A1 Cyanidin 3-O-galactoside
A2 Cyanidin 3-O-glucoside
A3 Cyanidin 3-O-arabinoside
A4 Cyanidin 3-O-xyloside
Total anthocyanins
Flavonols (360nm)
L5 Quercetin 3-O-rutinoside-7-O-glucoside
F1 Quercetin diglucoside
F2 Quercetin pentosilhexoside
F3 Quercetin 3-O-rhamnosyl-galactoside
F4 Quercetin 3-O-rutinoside
F5 Quercetin 3-O-galactoside
F6 Quercetin 3-O-glucoside
TOTAL FLAVONOLS
Flavones
L1 Apigenin 6,8-di-C-glucoside
L2 Diosmetin 6,8-di-C-glucoside
L3 8-C-glucosilchrysoeriol
L4 Diosmetin 7-O-rutinoside
Total avones
Hydroxycinnamic acid derivatives
C1 3-caffeoylquinic acid
L7 5-caffeoylquinic acid
L8 Ferulic acid
L9 Sinapic acid
Total hydroxicinnamic acids
Flavanones
L10 Eriodictyol-7-O-rutinoside
L11 Hesperetin-7-O-rutinoside
Total avanones

LA

40.84 0.04
1.87 0.00
16.51 0.01
2.03 0.01
61.25 0.06

39.97 2.02
1.90 0.02
16.33 0.32
2.16 0.15
60.36 2.21

nq

0.97 0.04

0.97 0.04

2.31 0.06
3.34 0.20
5.69 0.19

nq
1.61 0.01
3.22 0.12
5.02 0.11

6.76 0.69
3.73 0.29
21.82 1.64

5.14 0.15
2.83 0.08
18.57 0.46

1.10 0.01
6.24 0.05
nq
3.38 0.08
10.72 0.13

0.28 0.00
0.91 0.00
0.60 0.01
1.79 0.01

12.47 0.24

12.47 0.24

18.02 0.07
0.41 0.02
1.25 0.02
0.67 0.03
20.34 0.12

3.72 0.09
3.90 0.38
7.63 0.47

3.67 0.03
3.48 0.08
7.16 0.11

0.90 0.10
7.02 0.11
nq
4.64 1.64
12.55 1.85

Trolox/ml) (Bermdez-Soto & Toms-Barbern, 2004), as did dried

fruits, pomace, or other juices (279.38, 301.84, and 127.45 lmoles

Trolox/100 g dry weight, respectively) (Oszmianski & Wojdylo,

2005). The DPPH scavenging effect has been reported also for fruit

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A. Girons-Vilaplana et al. / Food Chemistry 134 (2012) 20902096

Table 3
Antioxidant and anticholinesterase (AChE and BuChE) activities of lemon juice, black
chokeberry, and lemon plus black chokeberry*.
DPPH
L
A
LA
LSD, p < 0.05

13.27
5.27b
4.23a
0.24

O2

HOCl
b

5.16
6.42c
3.24a
0.23

25.71
59.48c
28.95b
0.27

OH

AChE

BuChE

6.93c
4.90b
3.74a
0.17

13.18a
12.97a
10.57a
1.12

12.82b
18.98c
10.89a
0.44

IC50 values are expressed in mg/ml. L: Lemon juice, A: 5% black chokeberry

concentrate in citric acid control, LA: 5% black chokeberry concentrate in lemon
juice. Means (n = 3) in the same columns followed by different letters are signicantly different at P < 0.05 according to Tukeys test.

extracts (IC50 = 1.8 mg/ml, and 181.07 lmol TE/g) (Benvenuti et al.,
2004; Jakobek, eruga, Novak, & Medvidovic`-Kosanovic, 2007) and
for black chokeberry snacks (IC50 = 10.04 mg/ml) (GramzaMichaowska & Czapka-Matyasik, 2011), the latter being less
effective than the samples tested herein.
Previous reports described a direct correlation between the
DPPH scavenging activity and the total anthocyanins content of
anthocyanin-rich fruit extracts (Espn, Soler-Rivas, Wichers, & Garca-Viguera, 2000) and selected or red fruits (Dragovic-Uzelac, Levaj, Bursac, Pedisic, Radojcic & Biko, 2007; Jakobek et al., 2007). In
ve black chokeberry cultivars, a direct correlation between DPPH
scavenging and cyanidin 3-O-arabinoside and cyanidin 3-O-galactoside, also identied in our study, was found (Rop et al., 2010).
Nonetheless, no signicant correlation existed when the total avone glycosides of citrus varieties were compared with the DPPH
scavenging capacity (Xu, Liu, Chen, Ye, Ma & Shi, 2008). In accordance with these results, the higher activity of samples containing
black chokeberry concentrate (A, LA) can be related to their levels
of anthocyanins.
The controls (L and A) showed low values of IC50 for superoxide
(O2) scavenging assay, indicative of a high activity against this
radical. Moreover, the LA blend was more effective than the controls (Table 3). The polyphenols present in both lemon juice and
black chokeberry are potent O2 scavengers (Yu, Wang, Walzem,
Miller, Pike & Patil, 2005), including rutin, apigenin, and quercetin
(Masuoka, Matsuda, & Kubo, 2012). It is reasonable to suppose that
these avonoid glycosides with more than one en-diol group, like
quercetin or apigenin, chemically reduced this radical and decreased the superoxide generation (Masuoka et al., 2012). On the
other hand, anthocyanin extracts from rambutan (Nephelium lappaceum L.) or litchi (Litchi chinenesis Sonn.) showed excellent
superoxide anion scavenging activity (IC50 = 415.8 lg/ml and a
scavenging activity of 91.4% in a 50 lg/ml sample, respectively)
(Duan, Jiang, Su, Zhang, & Shi, 2007; Sun, Peng, Su, Yao, Long &
Wang, 2011). Scavenging activity of black chokeberry fruit against
the superoxide anion has been reported previously (Rop et al.,
2010). In summary, the anti-radical effect of the samples against
O2 may be due to their contents of non-coloured avonoids, such
as rutin, quercetin, or apigenin, and anthocyanins, since this effect
was more pronounced in the blend, which was richer in these
compounds.
The activity against HOCl was lower. The IC50 values varied signicantly, between 25.6 and 59.5 mg/ml (Table 3). The black
chokeberry control was far less active than lemon juice; hence,
the addition of black chokeberry concentrate to lemon juice did
not improve its activity (Table 3). Other work has demonstrated
that citrus pulp might be an effective HOCl scavenger (Ramful, Tarnus, Aruoma, Bourdon, & Bahorun, 2011) and showed a signicant
correlation between the total phenolic content of citrus extracts
and their scavenging of HOCl. This is consistent with the slightly
greater effect of lemon juice in comparison with the blends (Table
3). A weak protective effect against HOCl was described also for

cardoon (Cyanara cardunculus L.) infusion (Valento et al., 2002)

and beefsteak fungus (IC15 = 1.46 mg/ml) (Fistulina hepatica) extract (Ribeiro, Valento, Baptista, Seabra, & Andrade, 2007). No effect was found with Linaria vulgaris (Vrchovsk, Spilkov, Valento,
Sousa, Andrade & Seabra, 2008). However, other plant species
showed activity against the oxidative species; for example, Centarium erythraea (IC25 = 0.93 mg/ml) (Valento, Fernandes, Carvalho,
Andrade, Seabra & Bastos, 2003).
Regarding the hydroxyl radical (OH), the low IC50 values indicate good antioxidant capacity (Table 3). The black chokeberry
control displayed better activity than the lemon juice control, as
reported also for methanolic extracts of black chokeberry (Rop
et al., 2010). As was found for DPPH and O2-, the LA blend was
the most potent sample. Anthocyanin extracts from pomegranate
(Punica granatum) (Noda, Kaneyuki, Mori, & Packer, 2002) and
litchi (Duan et al., 2007) showed OH scavenging activity, mainly
due to chelation by metal ions rather than by direct scavenging
of the radical. However, ascorbic acid from lemon juice may act
as a pro-oxidant because of its ability to affect the redox cycling
of the metal ion required for the hydroxyl generation, thus increasing the radical production (Valento et al., 2002). The maximum
vitamin C content of black chokeberry was reported to be 30 mg/
100 g fw (Benvenuti et al., 2004; Skupien & Ozmianski, 2007),
but no vitamin C was found in our chokeberry samples, presumably owing to the thermal treatment used for the industrial concentration. Nevertheless, no pro-oxidant effect was observed in
the lemon juice, probably because the antioxidant potential of
the lemon avonoids offsets this effect.
So, the addition of black chokeberry concentrate to lemon juice
enhanced its antioxidant activity against the tested reactive species, except HOCl. The results suggest that the polyphenol content
is not the only reason for the antioxidant capacity; also, the quality
and the interactions between matter (Gramza-Michaowska &
Czapka-Matyasik, 2011).
3.3. Inhibition of AChE and BuChE
In recent years, the search for inhibitors of cholinesterases has
grown in interest, since these enzymes are associated with Alzheimers disease, senile dementia, ataxia, myasthenia gravis, and Parkinsons disease, among others (Mukherjee et al., 2007; Vinholes
et al., 2011). In vitro enzyme studies are used to screen for potential
bioactivity of certain substances against these enzymes, leading to
further studies in vivo. The IC50 values of the controls (L, A) and the
blend (LA) are presented in Table 3 and Fig. 1. All the samples displayed AChE and BuChE inhibitory activities, those of the lemon
juice and black chokeberry controls being similar for AChE. In the
BuChE assay, the lemon juice displayed a higher activity than the
concentrate (Table 3 and Fig. 1). The LA blend had the strongest
activity against both cholinesterases (Table 3 and Fig. 1). The avonols of lemon and black chokeberry, and the presence of C-glycosyl
avones in lemon, which have been demonstrated to be cholinesterase inhibitors (Khan et al., 2009), may have been responsible for
this. This effect was considerably lower than that of the pharmacological drugs used to treat neurological diseases, such as galantamine (IC50 for AChE = 0.14 lg/ml) (Wszelaki, Kuciun, & Kiss,
2010) or huperzine A (IC50 = 104 lM) (Mukherjee et al., 2007).
However, this new blend represents a natural alternative, which
can be taken every day, without side effects. It should be noted also
that it is not a puried extract. The IC50 values of other natural
products and extracts have been reported to range between 29
and 178 lg/ml (Wszelaki et al., 2010), while commercial oils
(IC50 = 0.0153.2 mg/ml; Dohi, Terasaki, & Makino, 2009) and tomato seeds (IC20 = 2.4 mg/ml; Ferreres et al., 2010) have been
tested also. Recently, the inhibition of AChE and BuChE by a
hydroxymethanolic extract of Spergularia rubra was tested

A. Girons-Vilaplana et al. / Food Chemistry 134 (2012) 20902096

2095

Fig. 1. Acetylcholinesterase and butyrylcholinesterase inhibitory activity of the samples: Lemon juice (L), black chokeberry control (A), and lemon juice plus 5% black
chokeberry concentrate (LA). Each point represents the mean of the values obtained from three independent experiments, in each of which triplicate samples were analysed.

(IC25 = 3.68 mg/ml and 4.29 mg/ml for AChE and BChE, respectively) (Vinholes et al., 2011), showing activity only slightly greater
than our samples.
Our results are very promising, because until now cholinesterase inhibitory activity of lemon juice or black chokeberry has not
been reported, as far as we know.
4. Conclusions
A new blend of lemon juice and black chokeberry concentrate
was characterised with respect to its functional activity and phytochemical prole. The results of different radical scavenging assays
indicate that the lemonblack chokeberry (LA) mixture (5% w/v,
black chokeberry concentrate) showed greater antioxidant capacity than the respective controls separately, except for HOCl, against
which lemon juice performed better. With respect to cholinesterases, both lemon and black chokeberry displayed inhibitory activity, which increased in the blend. Taking into account the role of
cholinesterases and their inhibition in neurodegenerative diseases,
these results are of interest regarding natural AChE and BuChE
inhibitors within foods. Therefore, this new blend has the potential
to be developed into a product with both nutritive and health promoting properties. Further metabolic and biological activity studies are necessary, as well as the search for more sources of
bioactive phytochemicals possessing these characteristics.
Acknowledgments
The authors express their gratitude to the Spanish Ministry of
Science and Innovation (MICINN) for funding through the project
C.I.C.Y.T. (AGL200761694/ALI) and the CONSOLIDER-INGENIO
2010 Research Project FUN-C-FOOD (CSD2007-00063). Part of this
work was carried out in international research collaboration within
the CYTED Programme (Ref. 112RT0460) CORNUCOPIA Thematic
Network. AGV thanks the CSIC for a JAE pre-doctoral grant, and
special thanks are due to the Department of Pharmacognosy in
Porto University for help in all the techniques employed for the
achievement of this work.
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